TCR-seq
Workflow
Calling Clonotypes
Calling method from contigs
1. Using Gene information
- VDJC(TRA/TRB)
- CDR3nt
- CDR3aa
- Combination
2. Using Chain information
- TRA1+TRB1
- TRA1 or TRB1
- multi-chains
Basic Clonotype Analysis
Immune repertoire analysis
1. Diversity Index
- Shannon Entropy
- Simpson
2. Repertoire analysis.
- TRAV/D/J/C distribution
- TRBV/D/J/C distribution
- CDR3nt length distribution
- Clustering of clonotype repertoire
3. Expansion study
- Clonotype expansion (hyper,high,medium,small,rare)
- Top 100 expanded clonotypes with TRAV:TRBV info
Target Cell:Clonotype Analysis
Combination with scRNA-seq (if available)
1. Target cell selection
- Cells with target features (e.g. cytotoxic cells, effector T cells, NKT, etc).
- Cells associated with expanded clonotypes (hyper, high, medium, small, rare).
- Cells with clonotypes displaying treatment-specific response.
2. Correlation of clonotype frequency with related data
- Correlation of clonotype frequency with target gene expression
profiles.
- Correlation with clinical outcomes and phenotypic data. (if available)
3. Comparative Analysis
- Comparison between different patient groups (e.g., responders vs non-responders to therapy, different disease stages) and associated clonotypes.
- Clonotype repertoires to identify conserved features of the immune response.
4. Statistical and Computational Approaches
- Application of statistical models and computational algorithms for
the analysis.
- Evaluation of the robustness and reproducibility of findings.
Example Plots
Clonotype Distribution
Clonotype Abundance
Sample A
Sample B
Cytotoxic Gene Expression Profiling across Clonotypes and Samples
Important Cytotoxic Gene Information
NKG7
- Natural Killer Growth Factor 7, the function of cytotoxic lymphocytes,
particularly Natural Killer (NK) cells, potentially through the
regulation of apoptotic pathways.
KLRG1
- Killer Cell Lectin-Like Receptor G1, is a transmembrane protein
receptor primarily expressed on cytotoxic lymphocytes, regulating their
activation and effector functions.
IFNG
- Interferon Gamma, is a cytokine crucially involved in the activation
and effector functions of cytotoxic lymphocytes, including NK cells and
cytotoxic T cells. IFNG enhances the cytotoxicity of these cells,
promotes antigen presentation, and directs T helper cell differentiation
towards a Th1 phenotype, crucial for effective immune responses.
GZMA
- Granzyme A, is a serine protease predominantly expressed in cytotoxic
lymphocytes, facilitating target cell apoptosis.Upon recognition of
target cells, GZMA is released and initiates caspase-independent cell
death pathways, leading to the elimination of infected or transformed
cells.
PRF1
- Perforin-1, is a pore-forming protein primarily expressed in cytotoxic
lymphocytes, essential for target cell lysis. PRF1 forms pores in target
cell membranes, enabling the delivery of pro-apoptotic molecules and
promoting target cell death, crucial for immune surveillance and
defense.
Heatmap